Upon neoplastic transformation there is an increase in aerobic glycolysis in certain types of cancer cells. In virus- transformed cell lines an elevated level of tyrosine protein kinase activity has been correlated with increased phosphorylation at tyrosine residues in several glycolytic enzymes. Although the cause and effect relationship between modification of the enzymes, rate of glucose metabolism, and the transformation phenotype is not clear, the correlation suggests that the alteration in the control of carbon flow through glycolysis might be involved in the transformation and or proliferation of cancer cells. In Saccharomyces cerevisiae mutations in GCR1 (Glycolysis Regulation) have a profound effect upon the level of most glycolytic enzymes. An understanding of the role of GCR1 in the regulation of glycolysis may provide insight into the regulation of glycolysis in higher eucaryotic cells and into the mechanism by which glycolytic regulation is altered in neoplastic cells. GCR1's mechanism of action is unknown, although an attractive model to consider is that GCR1 encodes a positive activator which specifically binds to the regulatory sequences of affected genes where it interacts with other proteins or factors to stimulate transcription of the corresponding structural gene. The major tenets of this model will be tested in the following manner: i) The half-lives of several affected RNA species will be determined in wild-type and mutant strains. Similar half-lives would indicate that GCR1 stimulates transcription. ii) Filter-binding and gel-retardation assays utilizing cloned DNAs encoding affected enzymes will be used to determine if GCR1 encodes an DNA binding protein. iii) DNaseI protection studies will be employed to define the DNA sequence recognized by the GCR1 gene product. iv) The GCR1- recognition sequence will be synthesized and cloned in front of a tester gene encoding beta-galactosidase. Then wild-type and gcr1 mutant strains will be used to assess the ability of GCR1 to effect expression of beta-galactosidase. v) Primer extension and nuclease S1 mapping techniques will be used to map the transcriptional start-sites of several affected genes in wild- type and mutant strains in order to determine if the gcr1 lesion reveals any transcriptional control features which are normally masked in wild-type strains. vi) Strains with gcr1-deletions will be used to isolated suppressors of gcr1 which occur spontaneously. Clones encoding suppressors will be characterized in order to identify other proteins with which GCR1 may interact to stimulate glycolytic gene expression.
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